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[Cancer Research 60, 936-943, February 15, 2000]
© 2000 American Association for Cancer Research

Endocrinology

The 4-Pregnene and 5{alpha}-Pregnane Progesterone Metabolites Formed in Nontumorous and Tumorous Breast Tissue Have Opposite Effects on Breast Cell Proliferation and Adhesion1

John P. Wiebe2, David Muzia, Juncai Hu, David Szwajcer, Scott A. Hill and Jennifer L. Seachrist3

Hormonal Regulatory Mechanisms Laboratory, University of Western Ontario, London, Ontario, N6A 5B7 Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
Progesterone is required for the full proliferative activity of the breasts and may be directly or indirectly involved in either stimulating or inhibiting breast cancer. To determine whether the effects on breast cancer are attributable to progesterone metabolites, we compared the capacity of nontumorous and tumorous breast tissue to convert progesterone and then tested the effects of these metabolites on breast cell proliferation and anchorage. Tissues from the operated breasts of six patients with infiltrating duct carcinomas were incubated with [14C]progesterone for 2, 4, and 8 h, and the metabolites were identified and quantified. The identified metabolites (equal to >95% of recovered radioactivity) can be divided into those that retain the double bond of progesterone in the carbon-4 position of ring A (4-pregnenes) and those that are 5{alpha}-reduced (5{alpha}-pregnanes). The results show that tumorous breast tissue has elevated 5{alpha}-reductase activity, which results in significantly higher total levels of 5{alpha}-pregnanes, especially 5{alpha}-pregnane-3,20-dione (5{alpha}P), whereas normal (nontumorous) breast tissue produces more 4-pregnenes, especially 3{alpha}-hydroxy-4-pregnen-20-one (3{alpha}HP). 5{alpha}P and 3{alpha}HP are each one enzymatic step removed from progesterone, resulting from the action of either 5{alpha}-reductase or 3{alpha}-hydroxysteroid oxidoreductase (3{alpha}-HSO), respectively. The ratio of 5{alpha}-pregnanes:4-pregnenes is >5-fold greater and the ratio of 5{alpha}P:3{alpha}HP is nearly 30-fold greater in tumorous than nontumorous breast tissue incubates. In vitro studies with three breast cell lines (MCF-7, MCF-10A, and ZR-75-1) show that 3{alpha}HP dose dependently inhibits, whereas 5{alpha}P significantly stimulates, proliferation. Additional studies with MCF-7 and MCF-10A cells indicate that each of the 4-pregnenes isolated from breast tissue suppresses, whereas each respective 5{alpha}-reduced product stimulates, cell proliferation. Studies of cell anchorage were conducted using MCF-7 cells and various concentrations of 5{alpha}P or 3{alpha}HP. The number of cells attached to the substrate was significantly (P < 0.05) decreased by treatment with >=30 nM 5{alpha}P and increased by treatment with >=50 nM 3{alpha}HP. Conversely, the number of cells detached from the substrate after partial trypsin exposure was significantly increased by treatment with >=40 nM 5{alpha}P and decreased by treatment with >=30 nM 3{alpha}HP. The results suggest that a change in in situ progesterone metabolism, resulting in an increased 5{alpha}-pregnane:4-pregnene (especially 5{alpha}P:3{alpha}HP) ratio, may promote breast cancer by promoting increased cell proliferation and detachment, whereas increases in 4-pregnenes may retard these tumorigenic processes. These studies suggest that endogenous progesterone metabolites may provide a new hormonal basis for breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
Potential roles for endogenous progesterone metabolites in human breast cancer appear to have not been investigated previously. It is well established that progesterone itself is involved in normal breast development as well as in the proliferative changes that occur in the breast during the menstrual cycle, pregnancy, and lactation (1–3) . A role for progesterone, and various derivatives (progestins) with progestational activities, in breast cancer treatment has been indicated by numerous reports. Thus, a number of studies have shown tumor regression resulting from progestin treatment (4, 5) at some doses, whereas in others, epithelial proliferation increased (6) . Retrospective studies suggest that surgery performed during the luteal phase of the menstrual cycle, when progesterone levels are higher, results in higher disease-free and overall survival rate than does surgery performed during the follicular phase (7–9) . In dogs, which have a very high incidence of spontaneous mammary cancers, administration of progesterone (but not estrogen) results in increased tumorigenesis (10) . In rodents, progestins may stimulate or decrease tumor growth (11) . Progestin treatments either increase or decrease growth of chemically [7,12-dimethylbenz(a)anthracene] induced tumors in vivo (12, 13) . Numerous in vitro studies on breast cancer cells have also shown that progesterone (or other progestins) can either stimulate or inhibit cell proliferation (14–17) and cell cycle progression (18–20) .

The dual, and seemingly paradoxical, actions of progesterone suggest the possibility that progesterone may be converted to two types of metabolites, those that stimulate and those that inhibit cell proliferation and tumorigenesis. The objectives of our studies were to identify and characterize the capacities of tumorous and nontumorous breast tissues to convert progesterone and then to test the presumptive active progesterone metabolites for their abilities to stimulate or inhibit cell proliferation and anchorage, functions that have been associated with breast cancer.

We report here the first evidence that tumorous breast tissue exhibits elevated 5{alpha}-reductase activity, which promotes significant increases in 5{alpha}-pregnanes, especially 5{alpha}P,4 whereas the normal (nontumorous) breast tissue produces more 4-pregnenes, especially 3{alpha}HP. In vitro studies with the breast cell lines MCF-7, ZR-75-1 (ER positive), and MCF-10A (ER negative) provide the first evidence that 3{alpha}HP and other 4-pregnenes inhibit, whereas 5{alpha}P and other 5{alpha}-pregnanes stimulate, breast cell proliferation and detachment. This is the first demonstration of the independent and opposing actions of the two classes of progesterone metabolites on cell proliferation and adhesion, two cardinal characteristics of cancer. The studies suggest that progesterone metabolites present a new endocrine link for breast cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
Chemicals and Biochemicals
[4-14C]Progesterone (57.2 mCi/mmol) was purchased from New England Nuclear (Dorval, Quebec, Canada) and was purified on TLC with chloroform:ether (10:3, v/v). Unlabeled 3{alpha}HP and 4-pregnene-3{alpha},20{alpha}-diol were prepared as described previously (21, 22) . Other unlabeled steroids were purchased from Steraloids (Wilton, NH) or Sigma Chemical Co. (St. Louis, MO). Progesterone and 3{alpha}HP were additionally purified by recrystallization. N-methyl-bis-trifluoroacetamide and N,O-bis(trimethylsilyl)trifluoroacetamide were obtained from Pierce Chemical Co. (Rockford, IL). BSA (fraction 5) and HEPES buffer were purchased from Boehringer-Mannheim Canada (Dorval, Quebec). Other chemicals and solvents were of appropriate analytic grade and were purchased from BDH, Inc., VWR, or Fisher Scientific Ltd. (Toronto, Ontario). Ethanol was glass distilled before use.

Human Tissues
Tissues from six patients (ranging in age from 44 to 84 years) were used in the study, and in each case, nontumorous and tumorous tissue was obtained from the same breast and used simultaneously in the metabolism studies. The tumorous tissues were diagnosed as infiltrating ductal carcinoma graded either as no. 2 or 3 on the Scarff-Bloom-Richardson histological grading system or as no. 4 on the modified Scarff-Bloom-Richardson (23) . Three tissues were positive for ERs and PRs, one was ER and PR negative, one was ER negative and PR positive, and one was ER positive and PR negative.

Breast Tissue Metabolism Studies
The tissues were frozen in liquid nitrogen immediately after surgery, transferred to the laboratory, and stored at -70°C until used for the studies (usually within 24 h). Each tissue was weighed immediately after removal from storage, homogenized with a Polytron P10 (Brinkman Instruments) at 23,500 rpm for 15 s in Krebs Ringer phosphate bicarbonate buffer (20% w/v). Aliquots of each homogenate (about 1.5 mg of protein/0.2 ml) were added to 0.5 ml of Krebs Ringer phosphate bicarbonate buffer containing [14C]progesterone (0.45 µCi/9.2 nmol) and NAD (0.75 mM), NADH (0.7 mM), NADP (2.6 mM), glucose-6-phosphate (7.7 mM), and glucose-6-phosphate dehydrogenase (Sigma Type XV, 1 unit). Incubations were carried out in glass tubes (16 x 100-mm) in a shaking water bath (60–80 cycles/min) at 37°C for 2, 4, and 8 h (two to three replicates each). One blank, containing all components except the tissue, was assigned to each incubation. Reactions were stopped by adding 5 ml of ether. The following standards (at 50 µg/50 µl methanol each) were added to each tube: 5{alpha}P, 5{alpha}P3{alpha}, 5{alpha}P3ß, 5{alpha}P20{alpha}, 4P20{alpha}, and 3{alpha}HP. For identification of 14C-labeled progesterone metabolites by GC/MS, incubations were carried out for 8 h with larger amounts of tissue and without addition of standards. In each case, the mixture was shaken for ~10 min, and the phases were separated either by freezing or by centrifugation. The ether phase was transferred to a clean extraction tube, and the aqueous phase was reextracted two more times with 5 ml of ether. The combined ether was evaporated under N2, the residue was brought up in 5–10 ml of 85% methanol (aqueous), and tubes were stored at -20°C. After 24 h, the liquid (unfrozen) solvent was transferred to fresh tubes, and the remaining lipid droplets were discarded. This delipidation process was repeated two more times. The methanol fraction was extracted three times with 5 ml ether, and the ether was evaporated under N2 at room temperature. Each extract was brought up in 1.0 ml of methanol, and an aliquot (5–10 µl) was used to determine the total radioactivity prior to chromatography. The methanol was evaporated under N2, and the residue was brought up in a small volume of dichloromethane.

Identification and Quantitation of Breast Tissue Progesterone Metabolites
TLC.
TLC was performed as described previously (24, 25) . Briefly, each extract was spotted on the lower right hand corner of a 20 x 20-cm silica gel G TLC plate (250 µm; Fisher Scientific, Pittsburgh, PA) previously activated at 100°C for 20 min. Each plate was run twice in solvent system 1 (chloroform:ether, 10:3, v/v) and then turned 90° and run three times in solvent system 2 (hexane:ethyl acetate, 5:2, v/v). Each plate was apposed to a sheet of Kodak Medical X-Ray film (X-OMAT R film) for 7–8 days before developing. Areas representing radioactive progesterone metabolites were marked on the TLC plates, and standards were visualized by exposure to iodine vapors. Each radioactive zone was scraped and extracted with a solution of ether:chloroform (4:1) to isolate individual steroids. The solvent was evaporated under N2, and the extract was brought up in 1.0 ml of methanol. Aliquots were used to determine the total radioactivity of each spot by scintillation spectrometry.

HPLC.
Aliquots of the TLC-separated metabolites were used in HPLC as described (25, 26) using UV and radioisotope detectors in series with a C18 column (Beckman, Ultrasphere ODS, 5 µm, 4.6 x 250-mm) and guard column (Ultrasphere ODS, 5 µm, 4.6 x 45-mm). For purposes of identifying radiolabeled metabolites, methanol:water at 3:1 (v/v) was used at a flow rate of 1 ml/min. Aliquots of the radiolabeled metabolites were injected, and retention times (Rt) of radioactive peaks compared with those of UV210 absorbance peaks (210 nm) of simultaneously run unlabeled steroid standards.

GC/MS Analyses.
GC/MS analyses were as described (25, 26) . Briefly, 14C-labeled metabolites (from extracts that did not contain unlabeled standards) were injected into a Hewlett-Packard GC-Mass Spectrometer (Models 5970A and 5790A GC and CHEMPC for Windows/DOS) with a 12.5-m (0.2-mm) cross-linked dimethyl silicone (HP Ultra-1) or a 30-m (0.25-mm) cross-linked 5% phenol methyl silicone (HP-5 MS) capillary column under conditions described previously (24, 26) . Retention times (on the capillary GC) and mass spectra were compared with those of the steroid standard(s) coinciding (on TLC and HPLC) with the unknown metabolite. In addition, computer-assisted comparisons were made with the mass spectra of ~350 different steroid standards, and their derivatives, on our HP GC/MS library. Preparation of derivatives by trifluoroacetylation (N-methyl-bis-trifluoroacetamide), trimethylsilyl ether formation or oxidation was as described (24) . Procedural losses were determined by quantifying the amount of added standard either by GC using a flame ionization detector or by HPLC (210 or 240 nm).


   Cell Lines and Culture
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
MCF-7 cells (passage #204; Karmanos Cancer Institute, Detroit, MI) were cultured in T-75 flasks (Falcon 3010) in phenol red-free DMEM-F12 Ham mixture (Sigma) containing L-glutamine and 15 mM HEPES and supplemented with 10 µg/ml bovine insulin, 5% (v/v) DCC-treated calf serum, 100 IU penicillin/ml, 100 µg of streptomycin/ml, and 10 µg/ml Fungizone under a humidified atmosphere of 95% air:5% CO2 at 37°C. The MCF-10A human breast epithelial cells (150th passage; Karmanos Cancer Institute) were cultured in T-75 flasks (Falcon 3010) similar to the MCF-7 cells with additional supplements of 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, and 0.5 µg/ml cortisone, until ~80% confluent. The ZR-75-1 human breast cancer cell line (83rd passage) was obtained from the American Type Culture Collection (Rockville, MD), and stock cells were routinely cultured in phenol red-free RPMI 1640 medium supplemented with 1 nM estradiol-17ß, 1 mM sodium pyruvate, 15 mM HEPES, 100 IU penicillin/ml, 100 µg streptomycin sulfate/ml, and 10% (v/v) DCC-treated fetal bovine serum under a humidified atmosphere of 95% air:5% CO2 at 37°C.


   Cell Growth Experiments
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
Stock cell cultures in late exponential growth phase (~80% confluent) were harvested and seeded in 24-well plates at about 4 x 104 cells/well and were allowed to attach for 24 h. Medium was removed, and cells were cultured in either control or treatment (with steroids) medium, supplemented as above, except that estradiol was omitted and that medium contained 5% (v/v) DCC-treated FBS (growth medium). Treatments were initiated (day 0) by replacing the medium with medium containing the indicated concentrations of steroids. Steroids were dissolved in 99% redistilled ethanol and added to the growth medium to final concentrations indicated in the results, whereas control cell cultures received the ethanol vehicle only (0.1% [v/v]), which had no detectable effect on cell growth and morphology. Medium changes occurred every 3 days or by daily replacement of 20% of the medium. Each treatment was replicated in five to six wells, and each experiment was repeated two to six times. Cells were harvested after 3 days of treatment or as indicated, and cell numbers were determined by a hemocytometer. [Some parallel experiments were also conducted using 96-well plates, and quantitation was via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method using a Dynatech MR600 microplate reader.]


   Cell Attachment and Detachment Assays
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
For the detachment studies, 10,000 MCF-7 cells were plated per 35-mm plastic (Falcon) dish (n = 4–8) and cultured for 72 h in DMEM-F12 medium as above (no estradiol) without (control) or with 5{alpha}P or 3{alpha}HP at various concentrations. Fresh media were added every 24 h. The media were removed, cells were rinsed with balanced salt solution and then exposed to trypsin (0.1% trypsin/0.05% EDTA in balanced salt solution) for 6 min at 23°C. After 6 min, 1.0 ml of medium without trypsin was added, and dishes were gently agitated on a rotary shaker (60 rpm) for 1 min. Media containing the detached cells were removed, and cell numbers were determined. The cell numbers remaining in the dishes were determined after further trypsinization. The number of detached cells was recalculated as the percentage of total number of cells/dish. For the attachment studies, cells were grown for 72 h in T-75 flasks without (control) or with 5{alpha}P or 3{alpha}HP at various concentrations. The cells were harvested and seeded at 10,000 cells per 35-mm dish (n = 4–8), and the number that remained unattached as well as those that had attached to the substrate after 2.5 h was determined using the trypsin/EDTA and gentle shaking regimen outlined above for the detachment studies.


   Statistics
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
Results are expressed as mean ± SE. Products formed as metabolites were expressed as ng/mg protein after corrections for procedural losses. Statistical analysis of the differences between nontumorous and tumorous tissue was performed by Student’s t test. The statistical significance of the effects of steroid treatments on cell proliferation and adhesion was determined using one-way ANOVA, followed by Tukey-Kramer Multiple Comparisons Test. Statistical calculations were performed using InStat software (GraphPad, San Diego, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 
Progesterone Metabolism by Nontumorous and Tumorous Breast Tissue
Breast tissue converted [14C]progesterone into at least 10 different metabolites (Fig. 1Citation ) 8 of which were definitively identified by TLC, HPLC, derivatization, and GC/MS. The metabolites can be divided into those that retain the double bond of progesterone in the carbon-4 position of ring A (4-pregnenes; nos. 2–6 in Fig. 1Citation ) and those that are 5{alpha}-reduced (5{alpha}-pregnanes; nos. 7–11 in Fig. 1Citation ). The amounts (per mg of protein) of the progesterone metabolites accumulated at the end of 2, 4, and 8 h of nontumorous and tumorous tissue incubations are presented in Table 1Citation .



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  		Fig. 1. Autoradiographs of TLC plates showing the products formed by incubating nontumorous (normal) and tumorous breast tissue from one patient with 15.36 µM (0.5 µCi) [14C]progesterone for 8 h. The radioactive spots and metabolites are identified as follows: spot 1, progesterone, {delta}4-pregnenes (4-pregnenes) 5{alpha}-pregnanes; spot 2, 3{alpha}HP; spot 3, 4P20{alpha}; spot 4, 4-pregnene-3,6,20-trione; spot 5/6, 4Pdiol, 4-pregnen-6{alpha}-ol-3,20-dione; spot 7, 5{alpha}P; spot 8, 5{alpha}P3{alpha}; spot 9, 5{alpha}P20{alpha}; spot 10, 5{alpha}P3ß; and spot 11, 5{alpha}Pdiol.

 

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  		Table 1 Progesterone metabolism by tumorous and nontumorous breast tissue

Tissues from six patients were used in the study, and in each case, nontumorous and tumorous tissue was obtained from the same breast. The tissues were frozen in liquid nitrogen immediately after surgery, transferred to the laboratory, and stored at -70°C until used for the studies (within 24 h). Each tissue was weighed immediately after removal from storage, homogenized in Krebs Ringer phosphate buffer (20% w/v), and incubated (37°C) with [14C]progesterone for 2, 4, and 8 h (generally three replicates each). Metabolites were extracted with ether and (together with appropriate standards) run two-dimensionally on TLC as described in "Materials and Methods." After autoradiography, the metabolites were identified by TLC, HPLC, derivatizations, crystallizations, and GC/mass spectrometry (23–25) and quantified by scintillation spectrometry after correction for procedural losses. Amounts of the progesterone metabolites at three incubation times (2, 4, and 8 h) are presented, following correction for procedural losses, as ng/mg protein (n = 6; mean ± SE).

 
In general, the combined amounts of all 5{alpha}-pregnanes produced by tumorous tissues were about 3–5 times as great as the amounts produced by nontumorous tissues; in contrast, the combined amounts of all 4-pregnenes were about 1.5-fold greater in the nontumorous than the tumorous tissue incubates (Fig. 2aCitation ). In nontumorous breast tissue, the 4-pregnene metabolites of progesterone greatly exceeded (by about 3.1–5.6-fold) the 5{alpha}-pregnanes (P < 0.001), whereas in tumorous tissue, 5{alpha}-pregnanes exceeded 4-pregnenes by about 1.4–1.65-fold. Thus, in the tissues from the six patients examined, there was approximately a 5–8-fold higher ratio of 5{alpha}-pregnane:4-pregnene metabolite quantities in tumorous than in nontumorous breast tissue, depending on incubation time. When values for different incubation times are combined, the average ratio of 5{alpha}-pregnane:4-pregnene amounts increased >5-fold, from ~0.3 in nontumorous to ~1.6 in tumorous breast tissues (Fig. 2bCitation ).



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  		Fig. 2. The difference in progesterone metabolism between nontumorous (Non Tum) and tumorous (Tum) breast tissue represented: a, as total mass (ng) of 4-pregnenes and 5{alpha}-pregnanes produced per mg of breast tissue protein; b, as the ratio of 5{alpha}-pregnanes:4-pregnenes; and c, as the ratio of 5{alpha}P:3{alpha}HP. Tumorous tissue exhibits nearly a 4-fold increase in 5{alpha}-pregnanes (a), more than a 5-fold increase in 5{alpha}-pregnane:4-pregnene ratio (b), and nearly a 30-fold increase in 5{alpha}P:3{alpha}HP ratio (c). Data from metabolism studies on six patients are shown. Columns, mean (from six patients) of all incubation times calculated from Table 1Citation ; bars, SE. *, significantly different from nontumorous tissue at P < 0.001.

 
The differences in 4-pregnene and 5{alpha}-pregnane amounts were mainly attributable to the two metabolites, 5{alpha}P and 3{alpha}HP (Table 1)Citation . On a per mg protein basis, 5{alpha}P comprised ~8–18% and 50–51% of the metabolites formed by the nontumorous and tumorous tissues, respectively, resulting in 3.9–8.0-fold higher values in tumorous than in the respective adjacent nontumorous part of the breast. On the other hand, 3{alpha}HP comprised 20–28% of the total of all metabolites formed by the nontumorous tissue and <4% by the tumorous tissue from each patient, resulting in 4.9–5.7-fold higher amounts in nontumorous tissues. In combined (2, 4, and 8 h) results, the mean ratio of 5{alpha}P:3{alpha}HP changed by nearly 30-fold, from 0.61 ± 0.16 in nontumorous to 18.16 ± 1.3 in tumorous breast tissue (Fig. 2cCitation ). Measurements of endogenous levels of 5{alpha}P and 3{alpha}HP by GC/MS in tissues from two patients confirmed elevated in situ levels of 5{alpha}P in tumorous breast tissue (results not shown).

Effects of Progesterone Metabolites on Breast Cell Proliferation
5{alpha}P and 3{alpha}HP.
The effects of the two progesterone metabolites, 3{alpha}HP and 5{alpha}P, the levels of which were most divergent between nontumorous and tumorous breast tissues, were examined with respect to cell proliferation in several breast cell lines.

MCF-7 Cells.
MCF-7 cells are ER positive, tumorigenic human adenocarcinoma cells. Treatment of MCF-7 cells (Fig. 3aCitation ) with 5{alpha}P resulted in significant, dose-dependent increases in cell numbers at 3, 5, and 7 days after the start of treatment, whereas 3{alpha}HP resulted in significantly fewer cells than in controls.



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  		Fig. 3. The dose- and time-dependent stimulatory and inhibitory effects of 5{alpha}P and 3{alpha}HP, respectively, on proliferation of MCF-7 (a), MCF-10A (b), and ZR-75-1 (c) breast cell lines. Data are the means of six separate experiments for a and b and one of two experiments for ZR-75-1 cells (c). Each point in an experiment had five to six replicates; bars, SE. The number of cells seeded/dish was 40,000 for a and b and 60,000 for c. Cells were exposed, for the time given, to no added steroid (Control) or to 10-8–10-6 M 5{alpha}P or 3{alpha}HP.

 
MCF-10A Cells.
To determine whether the response to progesterone metabolites is ER dependent and occurs also in nontumorigenic mammary cells, the effects on MCF-10A cells, a subline derived from human fibrocystic mammary tissue exhibiting characteristics of normal breast epithelium, lacking ER (27) , and tumorigenicity in nude mice, were examined. Treatment of MCF-10A cells (Fig. 3bCitation ) with 5{alpha}P resulted in significant, dose-dependent increases in cell numbers at 3, 5, and 7 days after the start of treatment, whereas 3{alpha}HP resulted in significantly fewer cells than in controls.

ZR-75-1 Cells.
The effects of 3{alpha}HP and 5{alpha}P on cell proliferation were also tested on ZR-75-1 cells, a mammary tumor cell line with receptors for estrogen, progesterone, and other steroid hormones and characterized to be human, malignant mammary epithelium in origin (28) . 5{alpha}P treatment resulted in significant dose- and time-dependent increases, whereas 3{alpha}HP treatment resulted in significant inhibition in ZR-75-1 cell number (Fig. 3cCitation ).

Other Progesterone Metabolites.
In light of the metabolic studies (Table 1Citation ; Fig. 1Citation ), which indicate a marked capacity of breast tissue to metabolize progesterone and to interconvert the metabolites, it became of interest to determine the activity, with respect to cell proliferation, of the other progesterone conversion products. In particular, we wanted to determine whether 5{alpha}-reduction of 4-pregnenes in general resulted in stimulation of cell proliferation. Treatment of MCF-7 and MCF-10A breast cell lines (Fig. 4Citation ) for 3 days with each of the 4-pregnenes identified in breast tissue incubates (i.e., progesterone, 3{alpha}HP, 4P20{alpha}, and 4Pdiol) resulted in significant inhibition of cell proliferation. In turn, each respective 5{alpha}-reduced product (5{alpha}P, 5{alpha}P3{alpha}, 5{alpha}P20{alpha}, and 5{alpha}Pdiol) resulted in stimulation of cell proliferation (Fig. 4a–hCitation ). By comparison, treatment with estradiol showed either slight stimulation of MCF-7 cell proliferation at low (10-8 M) concentrations or slight inhibition at 10-6 M (results not shown).



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  		Fig. 4. The opposing effects of {delta}-4-pregnenes and their respective 5{alpha}-reduced products on the replication of MCF-7 (a–d) and MCF-10A (e–h) breast cancer cells. The structures of the 4-pregnene and the 5{alpha}-reduced product are indicated at the top of each panel. Each 4-pregnene induced significant suppression of cell proliferation ({circ}). The respective 5{alpha}-pregnane resulting from the action of 5{alpha}-reductase induced stimulation of cell proliferation. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, SE.

 

   Effects of 3{alpha}HP and 5{alpha}P on Cell Adhesion
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
DISCUSSION
 REFERENCES
 
Carcinogenesis and tumor progression involve not only changes in cell proliferation but also alterations in cell adhesion properties. To determine the role of progesterone metabolites on cell anchorage, the effects of 5{alpha}P and 3{alpha}HP on MCF-7 cell adhesion were investigated. The percentage of cells that had either attached to, or detached from, the substratum after time and treatment conditions, as outlined in "Materials and Methods," was determined. Treatment of cells with 5{alpha}P for 3 days resulted in significant dose-dependent decreases in the percentage of cells attached (Fig. 5aCitation ) or increases in percentage of cells detached (Fig. 5bCitation ). Differences in cell attachment between control and 5{alpha}P-treated cells were significant (by ANOVA) at 30 nM and higher. Differences in cell detachment between control and 5{alpha}P-treated cells were significant at 40 nM and higher. On the other hand, treatment with 3{alpha}HP resulted in dose-dependent increases in cell adhesion (decreased detachment and increased attachment; Fig. 5Citation ). Treatment with 60 nM (and higher) 3{alpha}HP resulted in significant increases in the percentage of cells attached to the substrate. Treatment of cells with 20 nM (and higher) 3{alpha}HP resulted in significant (P < 0.05) decreases in the percentage of cells detached from the substratum.



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  		Fig. 5. The effect of 5{alpha}P (•) and 3{alpha}HP ({circ}) on MCF-7 cell attachment (a) and detachment (b) as a percentage of total number of cells. Hatched area, percentage of cells attached or detached in the controls (no steroids). a, for the attachment studies, cells were grown for 3 days in T-75 flasks without or with 5{alpha}P or 3{alpha}HP at various concentrations. The cells were harvested and seeded at 2 x 105 cells/dish (n = 4–8), and the number that had attached to the substrate after 2.5 h was determined. Significant (P < .05) decreases or increases, relative to the controls, in number of cells attached to the substrate resulted from >=30 nM 5{alpha}P or >=60 nM 3{alpha}HP, respectively. b, for the detachment studies, 5 x 104 cells were plated per 35-mm dish (n = 4–8) and allowed to culture for 72 h without (control) or with 5{alpha}P or 3{alpha}HP at various concentrations; the number of cells that had detached after a half-maximum trypsin exposure time was determined and is shown as a percentage of total number of cells. Significant (P < 0.05) increases or decreases, relative to the controls, in number of cells detached from the substrate resulted from >=40 nM 5{alpha}P or >=20 nM 3{alpha}HP, respectively. Bars, SE.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
REFERENCES
 
The hormonal environment in which mammary tumors develop and are maintained is important in understanding the progression of this disease. There is no convincing evidence that progressive changes in tumor development are related to circulating levels of steroids, and this may be because further metabolism of steroids occurs locally within the tumor and its adjacent host tissue. Such local metabolism might markedly change the biological potency of steroid hormones and create a microenvironment around and within tumor cells that promote the cancer.

Numerous studies have shown that breast tissue (tumorous and nontumorous) can produce estrogens from androgens (29–33) , dehydroepiandrosterone (34) , and estrogen sulfates (35) . Other studies have focused on the capacity of breast tissue to interconvert estrogens (36, 37) and to synthesize and metabolize catechol-estrogens (38) . The primary aim of these previous metabolism studies was to demonstrate estrogen synthesis and metabolism within the breast and to substantiate a link between estrogens (especially estradiol-17ß) and breast cancer. The metabolism of progesterone, on the other hand, has received little previous attention.

In the present study and in a previous study (39) , progesterone was shown to be metabolized by breast tissue. Lloyd (39) identified 5{alpha}P, 5{alpha}P3{alpha}, 5{alpha}P20{alpha}, 5{alpha}-pregnane-3{alpha},20{alpha}-diol, and 4P20{alpha} as metabolites of 3H-labeled progesterone. In using 14C-labeled progesterone, we were able to use autoradiography of two-dimensional TLC in visualizing essentially all of the radioactive metabolites. By these procedures, we have confirmed the production of the same metabolites found by Lloyd (39) and in addition have identified for the first time breast tissue conversion of progesterone into 3{alpha}HP, 4-pregnene-3{alpha},20{alpha}-diol, 4-pregnene-3,6,20-trione, and 4-pregnen-6{alpha}-ol-3,20-dione. On the basis of the metabolites identified, it is evident that human breast tissue has a number of progesterone-metabolizing enzymes including 5{alpha}-reductase, 3{alpha}-HSO, 3ß-HSO, 20{alpha}-HSO, 6{alpha}-hydroxylase, and 6{alpha}-HSO. From the results, it is evident that breast tissue can convert progesterone into two classes of metabolites: the {delta}-4-pregnenes (which retain the C4–5 double bond), and the 5{alpha}-reduced 21-carbon steroids (5{alpha}-pregnanes). The latter steroids are formed by the irreversible action of 5{alpha}-reductase. Each 4-pregnene (including progesterone) can be irreversibly reduced by the action of 5{alpha}-reductase to the respective 5{alpha}-pregnane metabolite. Within each class of metabolites, interconversions take place as a result of the reversible activities of the other enzymes. Fig. 6Citation illustrates this division of metabolites and illustrates the major interconversions within each class. Fig. 6Citation also shows that progesterone is converted by one enzyme step (involving 5{alpha}-reductase) into 5{alpha}P and by another (involving 3{alpha}-HSO) into 3{alpha}HP; progesterone can also be directly converted to 4P20{alpha} by 20{alpha}-HSO. Similar progesterone metabolic pathways have been demonstrated in ovarian (40) and pituitary (26) tissues.



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  		Fig. 6. The main pathways of progesterone metabolism in breast tissue. To the left are the {delta}-4-pregnenes (4-pregnenes), and to the right, the 5{alpha}-pregnanes and the respective interconversion enzymes within each pathway. Each of the 4-pregnenes (including progesterone) can be irreversibly converted by 5{alpha}-reductase to the respective 5{alpha}-pregnane. The two metabolites, 3{alpha}HP and 5{alpha}P, which show the greatest differences between tumorous and nontumorous tissue are indicated within the rectangle.

 
Our study showed quantitative differences between the normal and tumorous tissue metabolites. Of particular interest are the findings that in normal (nontumorous) breast tissue, the 4-pregnene metabolites of progesterone greatly exceeded the 5{alpha}-pregnanes, whereas in tumorous tissue, 5{alpha}-pregnanes exceeded 4-pregnenes. Thus, in the tissues from the six patients examined, there was approximately a 5–8-fold higher ratio of 5{alpha}-pregnane:4-pregnene metabolite quantities in tumorous than in nontumorous breast tissue. These differences in 5{alpha}-pregnane and 4-pregnene amounts were largely attributable to differences in 5{alpha}P and 3{alpha}HP production in tumorous and nontumorous tissues. These differences between tumorous and normal breast tissue are most dramatic when expressed as a ratio of 5{alpha}P:3{alpha}HP; the 5{alpha}P:3{alpha}HP ratio was about 0.6 in nontumorous and ~18 in tumorous breast tissue, resulting in a 30-fold increase in tumorous breast tissue. It should be noted that the metabolic activities were in general similar, regardless of the age and ER state of the patient or whether she was pre- or postmenopausal.

These findings suggest greatly elevated 5{alpha}-reductase activity in tumorous, as compared with nontumorous, breast tissue. The previous study by Lloyd (39) also showed elevated progesterone conversion to 5{alpha}-pregnanes formed by fibrocystic and carcinoma breast tissues. In rat mammary gland, after incubation with progesterone, 5{alpha}-reductase activity also was greater in malignant neoplasms than in normal tissue, and similar to our studies, 5{alpha}P was the principal metabolite (41) . In addition, 5{alpha}-reductase activity resulting in in vitro elevated conversion of testosterone to 5{alpha}-dihydrotestosterone has been reported for breast tissue in several studies (39 ; reviewed in Ref. 42 ).

If steroid metabolism within the breast is of biological importance, its role must be local within the breast itself. Even small conversions of inactive precursors into more active products might be associated with marked changes in the hormonal milieu within the breast. It may be that progesterone acts as a prohormone, and that metabolites such as 3{alpha}HP and 5{alpha}P may be important in regulating steroid-sensitive processes in breast tissue. 3{alpha}HP and 5{alpha}P (and other 5{alpha}-pregnanes) have been shown to exhibit important regulatory activities in the anterior pituitary, hypothalamus, and brain regions (43–48) . To determine whether progesterone metabolites have regulatory roles in breast cancer, we examined their effects on proliferation of breast cells in culture. It has been suggested (49) that of the several responses that the breast tissue shows to endogenous steroid hormones, the most reliable is that of cell proliferation. Many studies have provided evidence that estrogens may be mitogenic and play a role in breast cancer (reviewed in Ref. 3, 50 ). Although the role of progesterone in breast cancer has received less attention, there is now considerable evidence that progestins can influence the growth of target cells. Some studies suggest that progesterone is a proliferative hormone in the breast (3, 15, 20, 49–51 and references therein), and some report that progesterone exerts antiproliferative action (17, 20, 50, 52–54) .

Whether the actions attributed to progesterone may be attributable to its natural metabolites, to our knowledge, has not been examined previously. Our present studies provide the first evidence that progesterone metabolites that retain the C-4 double bond (i.e., the 4-pregnenes) exert an antiproliferative effect in the three cell lines that were tested, whereas the 5{alpha}-pregnanes stimulate breast cell line proliferation. The suppression of cell proliferation exerted by any one 4-pregnene tested was changed to stimulation when the 4-pregnene was reduced to its respective 5{alpha}-pregnane.

On the basis of this evidence, it is tempting to speculate that 4-pregnenes (such as 3{alpha}HP), by their antiproliferative actions, tend to hold cell proliferation in normal breast tissue in check, whereas 5{alpha}-pregnanes (such as 5{alpha}P), by their proliferative action, promote growth of the tumorous breast tissue. By this scheme, the degree of mitogenicity would be determined by the ratio of 5{alpha}-pregnanes:4-pregnenes. Tissues with a high 4-pregnene:5{alpha}-pregnane ratio would maintain a higher degree of normalcy, whereas those with a high 5{alpha}-pregnane:4-pregnene ratio would tend toward tumorigenicity. The observations that progesterone metabolites affect both ER-positive and ER-negative cells as well as tumorigenic (MCF-7) and nontumorigenic (MCF-10A) cells strengthen the argument that these factors may be endocrinologically relevant for all forms of breast cancer. Perhaps the apparent conflicting proliferative and antiproliferative actions suggested by the studies cited above may be, in part, explained by differential conversion of progesterone, by 5{alpha}-reductase activity, to either more or less 5{alpha}-pregnane products. The relative activity of 5{alpha}-reductase, therefore, may be important in breast cancer, just as it is in prostate cancer (55) .

In vitro, normal cells of either mesenchymal or epithelial origin usually depend on adhesion to, or spreading on, a solid substratum (anchoring) for cell division. As cells become neoplastic, they become less dependent on support of solid substrates for cell proliferation (56) . In vivo, these changes in adhesion that enable tumor cells to depart from the primary site of growth constitute the first step toward invasion and cancer metastasis. Therefore, the identification of endogenous factors that contribute to a change in cell adhesiveness is important to establish natural causes that prevent or promote the acquisition of metastatic potential. Having shown that the endogenous progesterone metabolites 5{alpha}P and 3{alpha}HP effect changes in cell proliferation, we tested their potential for altering adhesion (anchorage) in MCF-7 breast carcinoma cells. The results show that the metabolite 5{alpha}P, which caused significant stimulation of cell proliferation, also significantly decreased attachment (increased detachment) of cells from the substratum. The opposite effect resulted from the proliferation-inhibiting progesterone metabolite 3{alpha}HP, which promoted cell attachment and decreased cell detachment. Other studies have shown that various steroids, such as corticosteroids (57–59) , 1,25-dihydroxyvitamin D3 (60, 61) , androgens (62) , estrogens (63–65) , and progesterone (64, 66–68) may affect cell adhesion.

In the present context, it is of particular interest that estradiol, which is considered a mitogen in breast tissue, significantly suppresses adhesion of endometrial cancer cells (64) as well as MCF-7 cells (63) and inhibits endothelial vascular adhesion molecule-1 expression (65) . Progesterone may have similar (66–68) or opposite (64, 68) effects on cell adhesion. The opposing actions of 5{alpha}P and 3{alpha}HP on cell anchorage corroborate the proliferation effects and further strengthen the hypothesis that the direction of progesterone metabolism in vivo toward higher concentrations of 5{alpha}-pregnanes as opposed to 4-pregnenes might promote neoplasia. The mechanisms of action of 5{alpha}P and 3{alpha}HP on cell adhesion in terms of the cytoskeleton and adhesion complexes are being addressed.5

In summary, these results provide the first evidence that progesterone metabolites, rather than progesterone itself, may be linked to proliferative processes and cell adhesion in human breast cancer. Breast tissue is able to convert progesterone to two classes of steroids, the 4-pregnenes and the 5{alpha}-pregnanes. Each of the 4-pregnenes can be converted to a 5{alpha}-pregnane by means of the irreversible action of 5{alpha}-reductase. The production of 4-pregnenes (especially 3{alpha}HP) is greater in nontumorous breast tissue, whereas the production of 5{alpha}-pregnanes (especially 5{alpha}P) is greater in tumorous breast tissue. The 4-pregnenes significantly inhibit, whereas the 5{alpha}-pregnanes stimulate, proliferation and detachment of breast cell lines in vitro, thus exhibiting potent opposing actions on breast cells. The studies indicate that a change in in situ progesterone metabolism, resulting in an increased 5{alpha}-pregnane:4-pregnene (especially 5{alpha}P:3{alpha}HP) ratio, may promote breast cancer by stimulating increased cell proliferation and detachment, whereas increases in 4-pregnenes could retard these tumorigenic processes. These findings add a new concept to the hormonal control of breast cancer, which implicates the progesterone metabolites as the active endocrine/paracrine/autocrine factors. Estrogen-based therapies elicit responses in only one-third of all breast cancer patients, and most of these show relapse. Additional studies of the progesterone metabolites may offer the possibility of alternative endocrine approaches to the diagnosis and management of a wider range of breast cancers.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Canadian Breast Cancer Research Initiative Grant 007166. Back

2 To whom requests for reprints should be addressed, The University of Western Ontario, B&G Building, Room 344, London, Ontario, N6A 5B7 Canada. Phone: (519) 661-3131; Fax: (519) 661-2014; E-mail: jwiebe{at}julian.uwo.ca Back

3 Present address: Robarts Research Institute, Room 3.03-1, 100 Perth Drive, London, Ontario N6A 5K8, Canada. Back

4 The abbreviations used are: 5{alpha}P, 5{alpha}-pregnane-3,20-dione; 3{alpha}HP, 3{alpha}-hydroxy-4-pregnen-20-one; 4P20{alpha}, 4-pregnen-20{alpha}-ol-3-one; 4Pdiol, 4-pregnene-3{alpha}(ß),20{alpha}-diol; 5{alpha}P3{alpha}, 5{alpha}-pregnan-3{alpha}-ol-20-one; 5{alpha}P3ß, 5{alpha}-pregnan-3ß-ol-20-one; 5{alpha}P20{alpha}, 5{alpha}-pregnan-20{alpha}-ol-3-one; 5{alpha}Pdiol, 5{alpha}-pregnane-3{alpha}(ß),20{alpha}-diol; ER, estrogen receptor; PR, progesterone receptor; HSO, hydroxysteroid oxidoreductase; GC/MS, gas chromatography/mass spectrometry; HPLC, high-performance liquid chromatography; DCC, dextran-coated charcoal. Back

5 D. Muzia and J. P. Wiebe, manuscript in preparation. Back

Received 8/12/99. Accepted 12/16/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Cell Lines and Culture
 Cell Growth Experiments
 Cell Attachment and Detachment...
 Statistics
 RESULTS
 Effects of 3{alpha}HP and...
 DISCUSSION
 REFERENCES
 

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J. P Wiebe
Progesterone metabolites in breast cancer.
Endocr. Relat. Cancer, September 1, 2006; 13(3): 717 - 738.
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 J EndocrinolHome page
G. Perez-Palacios, R. Santillan, R. Garcia-Becerra, E. Borja-Cacho, F. Larrea, P. Damian-Matsumura, L. Gonzalez, and A. E Lemus
Enhanced formation of non-phenolic androgen metabolites with intrinsic oestrogen-like gene transactivation potency in human breast cancer cells: a distinctive metabolic pattern.
J. Endocrinol., September 1, 2006; 190(3): 805 - 818.
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 Endocr Relat CancerHome page
T. Suzuki, Y. Miki, Y. Nakamura, T. Moriya, K. Ito, N. Ohuchi, and H. Sasano
Sex steroid-producing enzymes in human breast cancer
Endocr. Relat. Cancer, December 1, 2005; 12(4): 701 - 720.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
Q. Ji, C. Aoyama, Y.-D. Nien, P. I. Liu, P. K. Chen, L. Chang, F. Z. Stanczyk, and A. Stolz
Selective Loss of AKR1C1 and AKR1C2 in Breast Cancer and Their Potential Effect on Progesterone Signaling
Cancer Res., October 15, 2004; 64(20): 7610 - 7617.
[Abstract] [Full Text] [PDF]

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 Cancer Epidemiol. Biomarkers Prev.Home page
C. H. van Gils, N. C. Onland-Moret, M. Roest, P. A. H. van Noord, and P. H. M. Peeters
The V89L Polymorphism in the 5-{alpha}-Reductase Type 2 Gene and Risk of Breast Cancer
Cancer Epidemiol. Biomarkers Prev., November 1, 2003; 12(11): 1194 - 1199.
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 EndocrinologyHome page
L. D. Griffin and S. H. Mellon
Biosynthesis of the Neurosteroid 3{alpha}-Hydroxy-4-pregnen-20-one (3{alpha}HP), a Specific Inhibitor of FSH Release
Endocrinology, November 1, 2001; 142(11): 4617 - 4622.
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 J. Clin. Endocrinol. Metab.Home page
T. Suzuki, A. D. Darnel, J.-I. Akahira, N. Ariga, S. Ogawa, C. Kaneko, J. Takeyama, T. Moriya, and H. Sasano
5{{alpha}}-Reductases in Human Breast Carcinoma: Possible Modulator of in Situ Androgenic Actions
J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2250 - 2257.
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